ORIGINAL PAPER
Description and genomic characterization of Streptococcussymci sp. nov., isolated from a child’s oropharynx
He Qi . Defeng Liu . Yang Zou . Nan Wang . Han Tian . Chunling Xiao
Received: 7 August 2020 /Accepted: 25 November 2020 / Published online: 2 January 2021
� The Author(s) 2021
Abstract Using the culturomics approach, we iso-
lated a new Streptococcus species, strain C17T, from
the oropharynx mucosa sample of a healthy 5-year-old
child living in Shenyang, China. We studied the
phenotypic, phylogenetic, and genomic characteristics
of strain C17T, which was identified as a Gram-
positive, coccus-shaped, non-motile, aerobic, cata-
lase-negative bacteria. Its growth temperatures ranged
from 20 to 42 �C, with optimal growth at 37 �C. Acidproduction could be inhibited by two sugars, trehalose
and raffinose. In C17T, the reactions for enzyme lipase
(C14) were confirmed to be negative, whereas those
for alkaline phosphatase, a-glucosidase, and hippuricacid hydrolysis were positive. The C17T genome
contained 2,189,419 base pairs (bp), with an average
G?C content of 39.95%, encoding 2092 genes in total.
The 16S ribosomal RNA sequence showed 99.8%
similarity with the newly identified Streptococcus
pseudopneumoniae ATCC BAA-960T. The main fatty
acid components in C17T were C16:0, C18:1 w7c,
C18:0, and C18:1 w9c, all of which can be found in
other species of the Streptococcus genus. Strain C17T
showed high susceptibility to clindamycin, linezolid,
vancomycin, chloramphenicol, and cefepime, and
moderate susceptibility to erythromycin. The obtained
dDDH value between strain C17T and the closest
species was 52.9%. In addition, the whole genome
sequence of strain C17T had an 82.21–93.40% average
nucleotide identity (ANI) with those strains of closely
related Streptococcus species, indicating that the strain
C17T was unique among all Streptococcus species.
Based on these characteristics, we determine that
C17T is a novel species, named Streptococcus symci
sp. nov. (= GDMCC 1.1633 = JCM 33582).
Keywords Culturomics � Human oral microbiota �Streptococcus symci �New species � Taxono-genomics
Introduction
As an important part of the salivary microbiome,
Streptococcus comprises 107 officially identified
species (https://lpsn.dsmz.de/genus/streptococcus),
which have been divided into six different groups
according to 16S ribosomal RNA (rRNA) sequence
H. Qi
Liaoning University of Traditional Chinese Medicine,
Shenyang, People’s Republic of China
H. Qi
Department of Medical technology, Medical Science
Institute of Liaoning, Shenyang, People’s Republic of
China
D. Liu � Y. Zou � N. Wang � H. Tian � C. Xiao (&)Key Lab of Environmental Pollution and Microecology of
Liaoning Province, Shenyang Medical College, No. 146,
Huanghe North Street, Shenyang, Liao Ning, People’s
Republic of China
e-mail: [email protected];
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Antonie van Leeuwenhoek (2021) 114:113–127
https://doi.org/10.1007/s10482-020-01505-3(0123456789().,-volV)( 0123456789().,-volV)
https://lpsn.dsmz.de/genus/streptococcushttp://crossmark.crossref.org/dialog/?doi=10.1007/s10482-020-01505-3&domain=pdfhttps://doi.org/10.1007/s10482-020-01505-3
results (anginosus, bovis, mitis, mutans, pyogenic, and
salivarius) (http://www.bacterio.net/) (Kawamura
et al. 1995). Many Streptococcus mitis strains are
highly virulent, which result in various pathologies,
such as meningitis, endocarditis, and pneumonia, by
invading the normal microbial community of low
pathogenic commensal species (Ricaboni et al. 2017).
In a previous study, four antagonistic Streptococcus
strains isolated from oropharyngeal microbiota were
found to have bacteriostatic effects on pathogens and
were involved in pharyngeal microbiome homeostasis
(Li et al. 2019). The pathogenic and commensal spe-
cies isolated from the upper respiratory tract of healthy
people exhibited similar morphology on culture
medium and were distinguished correctly and rapidly,
especially species sharing a 16S rRNA sequence
identity greater than 98.7%. This is of great signifi-
cance for screening probiotics and monitoring disease
epidemiology for clinical applications (Arbique et al.
2004). Therefore, it is necessary to use more accurate
and rapid methods for their identification. Generally,
the methods that are typically used include high-
throughput screening together with matrix-assisted
laser desorption/ionization-time of flight or 16S rRNA
sequencing to identify isolated colonies in order to
study the microbial community (Bittar et al. 2014).
Several different housekeeping genes have been
amplified for analysis, including sodA (Poyart et al.
1998), rpoB (Tapp et al. 2003), and groEL (Glazunova
et al. 2009), which are recognized as the classification
criteria for determining novel Streptococcus species
(Okamoto et al. 2015; Vela et al. 2015; Vela et al.
2016). DNA-DNA hybridization (DDH) is a key cri-
terion for the identification of new species (Auch et al.
2010). Average nucleotide identity (ANI) exhibits a
strong correlation with DNA-DNA hybridization
(DDH) values, with an ANI value C 95% corre-
sponding to the traditional 70% DDH threshold.
In this study, we analyzed the characteristics of a
novel species by using a series of cultivation and
genetic manipulations (Ramasamy et al. 2014) includ-
ing phenotype identification, housekeeping gene
sequencing, phylogenetic analysis, genome sequenc-
ing and annotation, fatty acid methylester analysis,
and the antibiotic susceptibility test. This novel
Streptococcus species was verified and named Strep-
tococcus symci C17T (= GDMCC 1.1633 = JCM
33582).
Materials and methods
Sample collection and strain isolation
In this study, pharyngeal swabs were used to collect
bacterial samples. Strain C17T was isolated from the
oropharynx mucosa sample of a healthy 5-year-old
child living in Shenyang, China in December 2015.
After sample collection, the pharyngeal swab was
immediately preserved in a 4 �C transport medium,sent to the laboratory, and stored at - 80 �C. Thebacterial samples obtained were dissolved in 1 mL
brain heart infusion (BHI) broth (bioMérieux, Cra-
ponne, France) and subsequently diluted to 1 L for
further experiments. Approximately 200 lL of thesample mixture was spread onto Columbia agar
(bioMérieux) supplemented with 5% (vv) defibrinated
sheep blood (Solarbio, Beijing, China) and incubated
at 37 �C aerobically in 5% CO2 for 24 h. Circularsingle colonies surrounded by a zone of a-hemolysiswere picked from the plates using an inoculation
needle, re-streaked on Columbia blood agar, and
incubated at 37 �C for another 24 h. Separate colonieswere chosen from the plate and cultured in liquid
medium until subsequent use.
Strain identification and gene sequencing of 16S
rRNA, groEL, rpoB, and sodA
Genomic DNA was isolated from the bacterial
colonies using the Wizard Genomic DNA Purification
Kit (Promega, Madison, WI, USA), and 16S rRNA
gene sequencing was conducted using the protocol
described by Delgado et al. (2006) and Jin et al.
(2013). The universal eubacterial primers 27F/1492R
(27F:50 - agagtttgatcmtggctcag -30 and 1492R:50 -ggytaccttgttacgactt -30) were applied during PCRanalysis using the Gene Amp PCR System 3730
Thermal Cycler (ABI, Vernon, CA, USA), as previ-
ously described (Drancourt et al. 2000). A nucleotide
basic local alignment search tool (BLASTn) analysis
(Altschul et al. 1990) was performed and aligned
within the national center for biotechnology informa-
tion (NCBI) database. Gene alignment results indi-
cated that the strain belonged to the Streptococcus
genus. The groEL, rpoB, and sodA genes of strain
C17T were amplified using the primer pairs strep-
togroELd/streptogroELr, 1730_F/3700_R, and d1/d2,
respectively, as previously described (Drancourt et al.
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114 Antonie van Leeuwenhoek (2021) 114:113–127
http://www.bacterio.net/
2004; Glazunova et al. 2009; Poyart et al. 2002).
Subsequently, BLAST analysis of these three genes
was performed using default NCBI parameters.
Phylogenetic analysis
The genome sequences of the Streptococcus genus
were obtained from the database list of prokaryotic
names with standing in nomenclature (http://www.
bacterio.net/streptococcus.html). The taxon of Strep-
tococcus is based on Bergey’s Manual of Systematics
of Archaea and Bacteria. The 16S rRNA genes of the
new Streptococcus isolates were sequenced, aligned
with those of other species of Streptococcus strains
and related taxa. Phylogenetic trees were constructed
and genetic distances were calculated using NCBI
analysis (https://www.ncbi.nlm.nih.gov/nucleotide/),
which was used for the sequence download of phylo-
genetically closest species. The sequences of groEL,
rpoB, and sodA from the closest species with standing
in nomenclature were directly downloaded from the
NCBI after BLASTn analysis. The phylogenetic tree
in this study was reconstructed with concatenated
groEL, soda, and rpoB sequences of strain C17T and
other closely related species. Alignment was per-
formed using MEGA X software (Tamura et al. 2013;
Kumar et al. 2018). The neighbor-joining method was
applied for phylogenetic inference generation. Boot-
strap analysis (1000 replications) was performed to
assess the reliability of the nodes.
Morphologic observation and optimal growth
conditions
After 24 h of incubation, the bacterial cells were
Gram-stained and observed using a Leica DM 500
photonic microscope (Leica Microsystems, Nanterre
Cedex, France) with a 100 9 oil immersion lens. Cell
morphology was determined using a scanning electron
microscope (Hitachi, Tokyo, Japan) set to the follow-
ing conditions: accelerating voltage 30,000 V, mag-
nification 7000, working distance 6700 lm, andemission current 112,000 nA. Cell motility was eval-
uated on soft agar plates (Xu et al. 2013). To determine
the optimal culture conditions, several culture condi-
tions were tested for strain C17T. Culture assays were
performed on Columbia agar supplemented with 5%
defibrinated sheep blood (bioMerieux) at temperatures
ranging from 4 to 45 �C (4 �C, 15 �C, 20 �C, 22 �C,
25 �C, 30 �C, 35 �C, 37 �C, 42 �C and 45 �C). Thesalt tolerance of strain C17Twas tested at various NaCl
concentrations (1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.5%
and 6.5%). The oxygen demand was tested under
aerobic, anaerobic, and microaerophilic (GENbag;
BioMerieux) conditions. Different pH values (from
4.0 to 10.0) were also tested. Hemolytic activity was
observed on Columbia blood agar plates. Catalase
assays (bioMerieux) were performed following stan-
dard protocols. The oxidase reaction was assessed
using the Becton Dickinson oxidase reagent (Becton
Dickinson, Franklin Lakes, NJ, USA).
Biochemical and fatty acid methylester analysis
and antibiotic susceptibility test
Biochemical analysis
The identification of API 50CH, API20 NE, and API
ZYM (bioMerieux) was used to distinguish Bacilli,
Enterococcus, and adjacent Streptococcus strains with
a positive enzyme test, and the experiments were
carried out according to standard instructions.
Fatty acid analysis
Each tube of samples was prepared using approxi-
mately 30 mg of bacterial biomass harvested from
several Columbia agar plates supplemented with 5%
sheep blood. Cellular fatty acids were then extracted,
modified, and analyzed according to the standard
protocol, using gas chromatography (Agilent 7890;
Agilent Technologies, Santa Clara, CA, USA) coupled
with the Sherlock Microbial Identification System
Version 6.3 (MIDI Inc., Newark, DE, USA).
Antibiotic susceptibility testing
The antibiotic susceptibility of strain C17T was tested
on antibiotic-sensitive paper (OXOID) using disk
diffusion assays following the Clinical Laboratory
Standards Institute 2018 recommendations. The
antibiotics used in this study were as follows:
clindamycin, 2 lg/mL; linezolid, 30 lg/mL; chloram-phenicol, 30 lg/mL; erythromycin, 15 lg/mL; cefe-pime, 30 lg/mL; vancomycin, 30 lg/mL; ampicillin,10 lg/mL; ceftriaxone, 30 lg/mL; and cefotaxime,30 lg/mL.
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Genomic DNA extraction, genome sequencing,
and assembly
Genomic DNA was extracted using the EZ1 DNA
Tissue Kit (Qiagen, Hilden, Germany) according to
the standard protocol. The DNA obtained was vali-
dated using gel electrophoresis and quantified using a
Qubit� 2.0 Fluorometer (Thermo Fisher Scientific,Waltham, MA, USA). Approximately 1 lg of totalDNA from each sample was used for sequencing.
Libraries for sequencing were constructed using the
NEBNext� UltraTM DNA Library Prep Kit forIllumina (New England Biolabs, Ipswich, MA, USA)
following standard recommendations, and index codes
were included to attribute sequences to each sample.
Each DNA sample was fragmented by sonication to an
average size of 350 base pairs (bp). The fragments
obtained were end-polished and A-tailed and then
ligated with the adapter for further PCR amplification.
Illumina PCR adapter reads and low-quality reads
were discarded after the quality control step using their
compilation pipeline. All paired-end reads with good
quality were assembled using the SOAP denovo online
software (Li et al. 2008; Li et al. 2010) (http://soap.
genomics.org.cn/soapdenovo.html) into different
DNA contigs that were handled by the next step for
gap closing. PCR products were purified (AMPure XP
PCR purification system; Beckman Coulter, Brea, CA,
USA), and libraries for size distribution were analyzed
with the Agilent 2100 Bioanalyzer and quantified
using quantitative PCR. The genome of C17T was
sequenced using the Illumina NovaSeq PE150 facility
(Illumina Inc., San Diego, CA, USA) at the Beijing
Novogene Bioinformatics Technology Co., Ltd.
(Beijing, China). Raw data were further processed in
four steps: discarding the reads of low-quality (BQ20)
bases and N-base to reach a certain proportion of reads
(default is 10%); discarding the reads whose overlap
with adapter exceeded a certain threshold value (de-
fault value is 15 bp) and mismatch number\ 3; andremoving adapter and duplication contamination.
Finally, 100 9 coverage of reads was obtained with
clean paired-end read data. The genome size was
estimated using k-mer statistical analysis before
assembly. Data were assembled with SOAP denovo
(version 2.04) and validated with SPAdes (Bankevich
et al. 2012) and ABySS (Simpson et al. 2009)
assemblers. Finally, the software CISA (Lin and Liao.
2013) was used for integration. Gap close (version
1.12) software was used to optimize and mend the
initial assembly results to obtain the final assembly
results. Fragments below 500 bp were filtered out.
Genome annotation and analysis
For the final assembled results of each sample to be C
500 bp, open reading frames were annotated using
Prodigal with standard settings (http://prodigal.ornl.
gov/) (Hyatt et al. 2010). The GeneMarkS program
(Besemer et al. 2001) (http://topaz.gatech.edu/
genemark/) was used to predict the coding region of
the newly sequenced genome. Transfer RNA (tRNA),
rRNA, and small nuclear RNA genes were analyzed
using tRNAscan-SE (Lowe and Eddy 1997),
rRNAmmer (Lagesen et al. 2007), and Pfam (Gardner
et al. 2009; Nawrocki et al. 2009) databases. The
interspersed repetitive sequences were analyzed using
Repeat Masker (Saha et al. 2008) (http://www.
repeatmasker.org/). Tandem repeats were analyzed
using a tandem repeats finder (Benson 1999). The
Island Path-DIOMB program (Hsiao et al. 2003) and
transposon PSI were used to predict the genomic
islands and transposons based on the homologous
BLAST method. Prophage prediction was carried out
by PHAST9 (Zhou et al. 2011) (http://phast.
wishartlab.com/), and clustered regularly interspaced
short palindromic repeat sequences (CRISPRs) were
identified using CRISPR Finder (Grissa et al. 2007).
The basic steps of the annotation function are listed
below: The predicted protein sequence was compared
with each functional database using diamond (e-value
B 1e-5). To filter the comparison results, the results
with the highest scores (default identity C 40%, cov-
erage C 40%) were selected for annotation. The bac-
terial proteome was predicted using the gene
prediction program GeneMarkS (version 4.28) toge-
ther with clusters of orthologous groups (COGs)
database. The Pfam (El-Gebali et al. 2019) database
was used to analyze protein function by identification
of PFAM-A and PFAM-B domains using the hhmscan
tool. The secreted proteins were predicted using the
Signal P database (Petersen et al. 2011), and the pre-
diction of Type I–VII proteins secreted by the patho-
genic bacteria was based on the Effective eT3
software (Eichinger et al. 2016). Meanwhile, the
secondary metabolism gene clusters were analyzed
using antiSMASH (Medema et al. 2011). To further
confirm the novelty of strain C17T, the genome-to-
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http://soap.genomics.org.cn/soapdenovo.htmlhttp://soap.genomics.org.cn/soapdenovo.htmlhttp://prodigal.ornlhttp://topaz.gatech.edu/genemark/http://topaz.gatech.edu/genemark/http://www.repeatmasker.org/http://www.repeatmasker.org/http://phast.wishartlab.com/http://phast.wishartlab.com/
genome distance calculator 2.1 (GGDC) was applied
to calculate the digital DDH (dDDH), which was
estimated with confidence intervals under the recom-
mended settings (Formula 2, http://ggdc.dsmz.de/
distcalc2.php).We also measured the overall similar-
ity among compared genomes by using Orthologous
Average Nucleotide Identity Tool ( Lee et al. 2015).
Results
Phylogenetic analysis
A comparative analysis of the 16S rRNA of strain
C17T showed a sequence identity of 99.8% with S.
pseudopneumoniae strain ATCC BAA-960T (Gen-
Bank Accession No. AY612844), 99.6% with S.
pneumoniae NCTC 7465T, and 99.4% with S. mitis
ATCC 494565T, which were the phylogenetically
closest species with standing in nomenclature (Fig. 1).
The concatenated comparison of sequenced gro EL,
rpoB, and sodA indicated that strain C17T and S. mitis
were in the same branch of the evolutionary tree and
had the most recent evolutionary relationship of all the
species of Streptococcus. This result also revealed that
the taxon represented by strain C17T was readily
distinguished from its nearest neighbors S. pseudop-
neumoniae strain ATCC BAA-960T and S. pneumo-
niaeNCTC 7465T (Fig. 2). The 16S rRNA sequence of
strain C17T was deposited in the GenBank with the
accession number MN068913.1.
Phenotypic characteristics and biochemical
features
Grass-green, a-hemolytic colonies of strain C17T wereobserved on 5% sheep’s blood-enriched Columbia
agar (bioMérieux) after 24 h of incubation under
aerobic conditions. Cells were confirmed to be Gram-
positive using classical staining (Fig. 3a) and cells
with a mean diameter of 5 lm (range, 4–8 lm) andnon-spore-forming rods (Fig. 3b) were observed using
scanning electron microscopy. The motility assay on
soft agar plates revealed the cells were non-motile.
C17T displayed a wide range of pH adaptability after a
growth test at different pH values (4.0, 4.5, 5.0, 5.5,
6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0). To
determine salt-tolerance ability, the growth of C17T
was observed in up to 2.5%NaCl. The growth of strain
C17T was also observed from 20 to 42 �C underanaerobic, microaerophilic, and aerobic conditions.
However, no growth was observed at 4 �C, 15 �C, or45 �C, and the optimal growth was found at 37 �Cunder aerobic conditions. Catalase and oxidase activ-
ity tests were negative. The identification and general
characteristics of strain C17T are summarized in
Table 1.
Strain C17T could be easily distinguished from the
nearest phylogenetic neighbors by its specific features,
and the biochemical profile of this novel species could
also be differentiated from those of closely related
species (Table 2), including 16S rRNA gene similar-
ity, lack of acid production from trehalose and
raffinose, negative reactions for lipase (C14), and
positive reactions for alkaline phosphatase, a-glucosi-dase, and hippuric acid hydrolysis. Using the API�20A strip (bioMérieux), positive reactions were only
observed for hippuric acid hydrolysis, leucyl-
aminopeptidase, and D-lactose fermentation. Negative
reactions were observed for the following tests: acid
production from starch, esculin hydrolysis, glycogen
hydrolysis, pyrrolidinyl arylamidase, Voges-Pros-
kauer reaction, a-galactosidase, b-galactosidase, b-glucuronidase, arginine hydrolase, and fermentation
of D-ribose, L-arabinose, D-mannitol, D-raffinose,
D-sorbitol, D-trehalose, L-arabinose, and inulin.
Using the API� ZYM strip (bioMérieux), positivereactions were observed as follows: alkaline phos-
phatase, esterase lipase (C8), trypsin, a-fucosidase, a-glucosidase, a-mannosidase, b-galactosidase, b-glu-curonidase, b-glucosidase, N-acetyl-b-glu-cosaminidase, and cystine arylamidase. Negative
reactions were observed as follows: acid phosphatase,
arylamidase, esterase (C4), leucine arylamidase,
lipase (C14), valine, a-chymotrypsin, a-galactosidase,and naphthol-AS-BI-phosphohydrolase. Using the
API� 50CH strip (bioMérieux), positive reactionswere observed as follows: D-galactose, D-fructose,
D-glucose, D-lactose, D-maltose, D-mannose, D-Su-
crose, and N-acetylglucosamine. Negative reactions
were observed as follows: D-adonitol, D-arabinose,
D-arabitol, D-cellobiose, D-fucose, D-lyxose, D-man-
nitol, D-melibiose, D-melezitose, D-raffinose, D-ri-
bose, D-saccharose, D-sorbitol, D-tagatose,
D-trehalose, D-turanose, D-xylose, methyl-a-D-glu-copyranoside, methyl-a-D-mannopyranoside, methyl-bD-xylopyranoside, amidone, amygdalin, arbutin,dulcitol, erythritol, esculin, gentiobiose, glycerol,
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Antonie van Leeuwenhoek (2021) 114:113–127 117
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glycogen, inositol, salicin, xylitol, L-arabinose, L-ara-
bitol, L-fructose, L-rhamnose, L-sorbose, L-xylose,
potassium gluconate, potassium 2-ketogluconate,
potassium, and 5-ketogluconate.
The main fatty acid components identified from
strain C17T were hexadecanoic acid (C16:0, 24.31%),
9-octadecenoic acid (C18:1 n9, 13.25%), branched
fatty acids (C18:1 n7/C18:1 n6, 13.16%), and octade-
canoic acid (C18:0, 12.39%), which could also be
detected in closely related Streptococcus species.
11-Hexadecenoic acid (C16:1 n5, 1.42%) was
detected in the isolated C17T strain rather than in
other types of closely related species. A complete fatty
acid analysis report of C17T and other related species
of the family Streptococcaceae are summarized in
Table 3.
In the antibiotic susceptibility test, strain C17T was
shown to be susceptible to clindamycin, linezolid,
vancomycin, chloramphenicol, and cefepime, and the
susceptibility of C17T to erythromycin was deter-
mined to be moderate. C17T was resistant to ceftriax-
one, ampicillin, and cefotaxime.
Genomic properties
The draft genome size of strain C17T was 2,189,419 bp
with a G?C content of 39.95% (Table 4, Fig. 4). It
contained eight contigs covering 2092 predicted genes
in total. Among these genes, 2057 were protein-coding
genes, and 43 were genes coding for RNAs (including
one 5S rRNA and 42 tRNA genes). A total of 340
genes were annotated as hypothetical proteins
(16.53%). A total of 1782 genes (85.18%) were
assigned to COGs, 201 of which were associated with
virulence (9.61%). To search for potential secondary
metabolite biosynthetic gene clusters (BGCs) in C17T,
Fig. 1 The phylogenetictree shows the distance
between S. symci strainC17T and other
Streptococcus strains. TheGen Bank accession number
of each strain is displayed in
brackets. Sequences were
aligned using ClustalW with
standard parameters.
Phylogenetic inferences
were generated using
MEGAX software following
the neighbor-joining method
with 1000 bootstrap
replicates and rooted using
E. faecalis JCM(AB012212) as the out
group. The scale bar
represents 1% nucleotide
sequence divergence
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118 Antonie van Leeuwenhoek (2021) 114:113–127
the genome sequence was unloaded to the antiSMASH
program (version 2.0.2) for detailed screening. Only
one BGC that was annotated as a bacteriocin was
found in C17T. Meanwhile, eight CRISPR repeats
were identified in the whole genome. Among the 25
general COG functional categories, five were not
assigned to eight closely related species, including
RNA processing and modification, chromatin struc-
ture and dynamics, nuclear structure, and cytoskele-
ton. Eight COG functional categories were grouped
with more associated genes in C17T than other closely
related strains. The detailed distribution of genes was
as follows: translation, 164 genes; amino acid trans-
port and metabolism, 161 genes; cell wall/membrane
biogenesis, 121 genes; nucleotide transport and
metabolism, 73 genes; posttranslational modification,
protein turnover, and chaperons, 71 genes; signal
transduction mechanism, 56 genes; energy production
and conversion, 52 genes. The genome statistics are
presented in Table 4, and the gene distribution into
COG functional categories is summarized in Table 5.
Fig. 2 groEL, rpoB, andsodA-based phylogenetictree showing the position of
S. symci strain C17T relativeto other Streptococcusstrains. The Gen Bank
accession numbers of the
strains are shown in
brackets. Sequences were
aligned using ClustalW with
default parameters.
Phylogenetic inferences
were obtained using
MEGAX software following
the neighbor-joining method
with 1000 bootstrap
replicates. The scale bar
represents 1% nucleotide
sequence divergence
Fig. 3 Phenotypic features of S. symciC17T aGram-staining of S. symciC17T. b Scanning electronmicroscopy image of S. symciC17T
using S-3400N (Hitachi Company) at an operating voltage of 30 keV. Scale bar = 5 lm
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Antonie van Leeuwenhoek (2021) 114:113–127 119
Genomic comparative analysis between C17T
and closely related species
To calculate the dDDH between C17T and other
available species that are phylogenetically closest
(Table 6), the GGDC online formula 2 calculator was
used for detailed comparative analysis. Strain C17T
displayed dDDH values of 47.20, 52.90, 30.90, 44.90,
and 26.20 for S. pseudopneumoniae ATCC BAA-
960T, S. mitis ATCC 49456T, S. oralis ATCC 35037T,
S. pneumoniae NCTC 7465T, and S. infantis ATCC
15192T, respectively. These dDDH values were lower
than the threshold value of 70% for species demarca-
tion. The pair-wise ANI values between strain C17T
and the type strain of other Streptococcus species were
91.99%, 93.40%, 85.90%, 91.42% and 82.21%
respectively, thereby indicating that the newly isolated
strain is representative of a new Streptococcus species.
The distribution of the predicted genes of S. symci
C17T to different COG functional categories is
summarized in Fig. 5.
Discussion
Recognized as an important part of commensal
microbiota in humans, Streptococcus species are
widely distributed in all parts of the human body,
especially the mouth, skin, intestine, and upper
respiratory tract. They are responsible for many types
Table 1 Classification and general features of strain Strepto-coccus symci C17T
Property Term
Classification Domain Bacteria
Phylum Firmicutes
Class Bacilli
Family Streptococcaceae
Genus Streptococcus
Species Streptococcus symci
Type strain C17T
Gram stain Positive
a-Hemolytic Positive
Cell shape Cocci
Motility Non-motile
Sporulation Non-spore forming
Oxygen requirement Aerobe
Temperature range 22–42 �COptimum temperature 37 �CSalt tolerance \ 2.5%Catalase Negative
Oxidase tested Negative
Biotic relationship Free-living
Origin Respiratory tract of a healthy child
Table 2 Biochemical characteristics used to differentiateStreptococcus symci C17T from the nearest neighbor species ofStreptococcus
1 2 3 4 5 6 7
API ZYM
Alkaline Phosphatase ? - - - - ? ?
b-Galactosidase V - ? ? ? - ?
a-Glucosidase ? NA - - - - -
b-Glucosidase ? - v - - ? -
Lipase (C14) - NA v ? ? ? v
API 20 Strep
Voges–Proskauer test - - - - - - -
Arginine - v - - - - ?
Esculin - - - V - - -
Hippuric acid ? - - - - - -
Pyrrolidinyl arylamidase - NA ? - - - -
a-Galactosidase - ? - - - ? -
Ribose - v v - - - -
Mannose - ? - - - - -
D-raffinose - v v - ? ? -
Starch - v - ? - - -
API 50CH
D-galactose ? ? NA ? ? ? ?
D-glucose ? ? NA ? ? ? ?
D-fructose ? ? NA ? ? ? ?
Amygdalin - - NA - ? - -
D-mannitol - v - - - - -
D-sorbitol - - - - - - -
Melibiose - v - v - - -
D-tagatose - - - - - - -
D-lactose ? ? ? ? ? ? ?
D-sucrose ? ? V ? ? ? ?
D-trehalose - V - ? ? - -
Strains: 1, S. symci C17T; 2, S.mitis ATCC 49456T; 3, S.pseudopneumoniae ATCC BAA-960T; 4, S. oralis ATCC35037T; 5, S. infantis ATCC 15192T; 6, S. dentisani DSM27089T; 7, S. australis ATCC 700641T. ?, positive reaction;-, negative reaction; V, variable; NA, data not available
123
120 Antonie van Leeuwenhoek (2021) 114:113–127
of diseases, including meningitis, pneumonia, and
erysipelas (Krzysciak et al. 2013). However, many
Streptococcus species are nonpathogenic symbionts.
Here, we isolated a Streptococcus strain C17T from the
oropharynx mucosa sample of a healthy 5-year-old
child. Phenotypic and biochemical feature
Table 3 Cellular fatty acid composition (%) of C17T and other closely related species
Fatty acids Name 1 2 3 4 5 6
C16:0 Hexadecanoic acid 24.31 35.5 31.45 36.54 32.34 34.2
C18:1 n9 9-Octadecenoic acid 13.25 11.35 12.75 10.59 11.05 14.86
Sum In Feature 8 18:1 n7/18:1 n6 13.16 7.43 10.02 6.97 6.51 5.96
C18:0 Octadecanoic acid 12.39 12.52 12.35 11.61 11.02 12.82
Sum In Feature 5 18:2 n6, 9/18:0 anteiso 8.92 6.29 7.41 5.46 7.77 7.35
Sum In Feature 3 16:1 n7/16:1 n6 8.54 3.12 1.55 3.15 3.57 2.89
C16:1 n9 7-Hexadecenoic acid 6.46 1.1 3.75 1.5 2.75 1
C14:0 Tetradecanoic acid 6.42 14.02 9.95 15.21 14.85 11.47
C12:0 Dodecanoic acid 1.48 4.95 TR 4.97 4.24 4.31
C16:1 n5 11-Hexadecenoic acid 1.42 TR TR TR TR TR
C20:4 n 6, 9, 12, 15 5, 8, 11, 14-Eicosatetraenoic acid, etc. 1.19 TR 1.12 TR 1.19 TR
C17:0 Heptadecanoic acid 1.00 TR TR TR 1 1.05
C17:0 anteiso 14-Methyl-hexadecanoic acid ND TR TR TR TR TR
C20:1 n9 Cis-11-Eicosenoic acid ND ND 4.19 ND ND ND
C15:0 Pentadecanoic acid TR TR 1.34 TR 1.08 1.24
C15:0 anteiso 12-Methyl-tetradecanoic acid TR ND ND TR ND ND
C17:0 iso 15-Methyl-Hexadecanoic acid ND ND ND TR TR ND
C13:0 Tridecanoic acid ND ND ND TR TR TR
C18:1 n5 13-Octadecenoic acid ND ND TR TR ND ND
Strains: 1, S. symci C17T; 2, S. oralis ATCC 35037T; 3, S. infantis ATCC 15192T; 4, S.dentisani DSM 27089T; 5, S.australis ATCC700641T; 6, S. pseudopneumoniae ATCC BAA-960T. ND: not detected; TR: trace amounts\ 1%.
Table 4 Nucleotide content and gene counts of the genome of S.symci C17T
Attribute Genome
Number Total percentage (%)
Genome Size (bp) 2,189,419 100
G?C Content 874,673 39.95
Total number of genes 2092 100
Total number of protein-coding genes 2057 98.33
Total number of RNA Genes 43 2.06
Total number of tRNA Genes 42 2.01
Total number of rRNA (5S, 16S, 23S) Genes 1 0.05
Coding sequence gene protein size (bp) 1981,482 90.50
Number of proteins associated with clusters of orthologous groups 1782 85.18
Number of proteins with peptide signal 88 4.21
Number of genes associated with virulence 201 9.61
Number of proteins with transmembrane helix 557 26.63
Genes associated to bacteriocin 12 0.57
123
Antonie van Leeuwenhoek (2021) 114:113–127 121
identification, phylogenetic analysis, and genome
annotation were performed. The results indicated that
C17T was a new species of the Streptococcus genus.
Set as a key criterion, a 70% threshold of dDDH
value has been adopted to delimitate a species (Auch
et al. 2010; Meier-Kolthoff et al. 2013; Wayne 1988).
The dDDH values of C17T with other adjacent strains
calculated by GGDC (online formula 2 calculator)
were all less than 70%. Among all the comparative
analyses, the dDDH for estimating the genomic
distance between strain C17T and the nearest S.
pseudopneumoniae (16S rRNA closest species with
standing in nomenclature) was 47.2%, while the value
for estimating C17T compared with S.mitis (Gro EL,
rpoB, and sodA genes) was 52.9%. For the compar-
ative analysis of C17T with other Streptococcus
species, the dDDH values were even lower, 30.9%
for S. oralis ATCC35037T, 44.90% for S. pneumoniae
Fig. 4 The genome graphical circular map of S. symci C17T.The outermost circle is the position coordinate of the genomic
sequence. From outer to inner: coding DNA sequences on the
forward strand (the outer chain), coding DNA sequences on the
reverse strand (the inner chain), COG category of genes on the
forward strand (the positive chain by the outer circle); COG
category of genes on the reverse strand (the negative chain by
the inner circle); genome GC content (inward red part indicates
that the GC content in this area is lower than the whole genome
average GC content, the outward green part is opposite),
genomic GC skew value (pink part indicates that the area G
content is lower than C Content, the outward light green part is
opposite). (Color figure online)
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122 Antonie van Leeuwenhoek (2021) 114:113–127
NCTC7465T, and 26.20 for S. infantis ATCC15192T
(Table 6).The genome sequence of strain C17T had
82.21–93.40% ANI with type strains of other Strep-
tococcus species (Table 7), which are below the C
95% ANI cut-off to define a bacterial species (Richter
and Rossello-Mora 2009). Thus, the results of genome
distance analysis provide strong evidence supporting
the identification of S. symci C17T as a new Strepto-
coccus species. The results of phenotype analysis
obtained through API strips indicated that strain C17T
possessed unique profiles of enzyme spectra and sugar
utilization for fermentation, compared with other
closely related species (Table 2). The biochemical
features of other neighboring strains were consistent
with those reported in the literature (Huch et al. 2013).
The fatty acid composition of C17T was also clearly
distinct from other closely related species, indicating
the uniquemetabolome profile of C17T. 16S rRNA can
only be used for strain identification for classification
up to the genus level; thus, among all Streptococcus
species, the similarity of C17T to other Streptococcus
species with highly homologous 16S rRNA was a
common feature (Fig. 1). Meanwhile, the gene com-
parison analysis of concatenated groEL, rpoB, and
sodA demonstrated high sequence identity with the
closest S.mitis strain ATCC 49456T (Fig. 2). This
result was consistent with that of the DDH analysis.
The genomic analysis of C17T showed that eight COG
Table 5 Number of genes associated with the 25 general clusters of orthologous group functional categories
Code Description S.symci
S.
mitisS.oralis
S.dentisani
S.pneumoniae
S.pseudopneumoniae
S.infantis
S.tigurinus
J Translation 164 146 147 146 0 0 0 0
A RNA processing and modification 0 0 0 0 0 0 0 0
K Transcription 111 116 101 106 I28 129 91 126
L Replication, recombination and repair 117 111 94 115 199 153 112 157
B Chromatin structure and dynamic 0 0 0 0 0 0 0 0
D Cell cycle control, mitosis and meiosis 21 19 23 20 21 21 23 21
Y Nuclear structure 0 0 0 0 0 0 0 0
V Defense mechanisms 70 46 40 53 72 81 47 58
T Signal transduction mechanism 56 52 45 50 46 55 46 49
M Cell wall/membrane biogenesis 121 95 95 91 118 89 105 101
N Cell motility 3 1 3 2 1 1 2 2
Z Cytoskeleton 1 0 0 0 0 0 0 1
w Extracellular structures 1 II n II II II II 1
U Intracellular trafficking and secretion 27 20 30 25 25 22 22 37
O Post translational modification, protein
turnover, chaperones
71 59 62 53 65 62 52 59
C Energy production and conversion 52 48 41 41 49 41 40 46
G Carbohydrate transport and 129 130 112 125 194 152 111 140
E Amino acid transport and metabolism 161 137 145 155 154 147 I28 142
F Nucleotide transport and metabolism 73 65 68 68 65 67 65 70
H Coenzyme transport and metabolism 48 41 41 43 43 50 36 43
I Lipid transport and metabolism 32 30 30 31 31 30 30 33
P Inorganic ion transport and 100 93 90 88 114 101 76 94
Q Secondary metabolites biosynthesis,
transport and Catabolism
13 10 11 11 12 12 11 15
R General function prediction only 0 0 0 0 0 0 0 0
S Function unknown 412 339 384 377 384 406 380 440
123
Antonie van Leeuwenhoek (2021) 114:113–127 123
functional categories were distributed with more
associated genes in C17T, compared with the other
closely related species, indicating that C17T is differ-
ent from the other known Streptococcus species at the
genetic level.
In this study, the preliminary tests showed that
strain C17T had the effect of antagonizing pathogens,
which was different from that of S. mitis. Together
with other studies on strain characteristics, it was
concluded that the classification of strain C17T was
Table 6 The dDDH values (%) obtained by a comparative analysis of Streptococcus symci C17T and other closely related species(calculated by GGDC formula 2, DDH was estimated based on identity/HSP length)
S.symci S. pseudopneumoniae(%)
S. mitis (%) S. oralis (%) S. pneumonia(%)
S. infantis (%)
S. symci 100 47.20 (44.6–49.8) 52.90(50.3–55.6)
30.90
(28.5–33.4)
44.90
(42.4–47.5)
26.20
(23.9–28.7)
S.pseudopneumoniae
100 48.20
(45.6–50.8)
31.80
(29.4–34.3)
58.80 (56–61.6) 26.40
(24.1–28.9)
S. mitis 100 31.70(29.3–34.2)
46.30
(43.7–48.8)
26.10
(23.7–28.6)
S. oralis 100 31.50(29.1–34.1) 29.60(26.7–32.7)
S. pneumoniae 100 25.90(23.5–28.3)
S. infantis 100
Fig. 5 Distribution of the predicted genes of S. symci C17T to different COG functional categories, compared to other closely relatedspecies
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124 Antonie van Leeuwenhoek (2021) 114:113–127
similar, but still did not belong to S. mitis. Unfortu-
nately, due to the high genomic similarity with various
pathogenic Streptococcus genera, we still have no
adequate evidence to rule out the possibility that this
bacterium is virulent, and no virulence factor analysis
was conducted in this study. However, strain C17T is
an independent species in the Streptococcus genus and
may develop into a type of probiotic that is necessary
for maintaining the health of the human oropharynx.
Description of S. symci sp. nov.
Streptococcus symci (sym’ci. N.L. gen. n. symci,
arbitrary epithet derived from Shenyang Medical
College, where the sample was characterized).
It is a non-motile, non-spore-forming, aerobic, and
Gram-positive bacteria with an approximate diameter
of 5 lm. The cells formed grass-green, a-hemolyticcolonies on Columbia agar plates containing 5% sheep
blood after 24 h of incubation. The cells can grow at a
temperature ranging from 20 to 42 �C with an optimaltemperature of 37 �C under anaerobic, microaerophi-lic, and aerobic conditions. The growth of C17T was
observed at different pH values (from 5.0 to 8.5) and
salt concentrations of up to 2.5% NaCl. No oxidase
and catalase activities were detected. The major fatty
acids were hexadecanoic acid (24.31%), 9-octade-
cenoic acid (13.25%), branched fatty acids C18:1 n7/
C18:1 n6 (13.16%), and octadecanoic acid (12.39%).
This strain of Streptococcus symci sp. nov., C17T,
was first isolated from the oropharynx of a healthy
5-year-old child in Shenyang. The size of the genome
was 2,189,419 bp, with a DNA G?C content of
39.95%. The 16S rRNA sequences of C17T were
uploaded to the GenBank with the accession number
MN068913.1. The whole-genome shotgun sequence
was uploaded to the GenBank under accession number
VFJA00000000. The habitat of this bacterium was a
healthy oropharynx.
Author contributions CX: Conceptualization (lead); HQ:Resources (lead); Data Curation (lead);Formal Analysis
(lead);Writing–original draft (lead); Writing–review and
editing (lead); DL: Methodology (equal);Formal Analysis
(equal);Writing–original draft (equal); YZ: Methodology
(equal); Formal Analysis (equal);Writing–original draft
(equal); NW: Methodology (equal);Resources (equal);Data
Curation (equal); HT: Methodology (equal); Validation
(equal); Writing–original draft (equal). All authors read and
approved the final manuscript.
Funding This study was supported by the Shenyang Scienceand Technology Bureau Project (18-400-09).
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
Ethics approval The study was conducted in accordance withthe guidelines of the ‘‘Helsinki Declaration’’ and approved by
the ethics committee of Shenyang Medical College under
number 2015052902.
Informed consent The participating donors providedinformed consent.
Open Access This article is licensed under a CreativeCommons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit
to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are
included in the article’s Creative Commons licence, unless
indicated otherwise in a credit line to the material. If material is
not included in the article’s Creative Commons licence and your
intended use is not permitted by statutory regulation or exceeds
Table 7 ANI values (%) between S. symci C17T and other closely related species (calculated by the OrthoANI algorithm)
S. symci S. pseudopneumoniae (%) S. mitis (%) S. oralis (%) S. pneumonia (%) S. infantis (%)
S. symci 91.6712 93.1873 85.3982 90.8849 81.5349
S. pseudopneumoniae 81.4333 81.3882 81.2183 81.6869
S. mitis 85.76 91.4778 92.1375
S. oralis 85.4564 85.6727
S. pneumoniae 94.44
S. infantis
123
Antonie van Leeuwenhoek (2021) 114:113–127 125
the permitted use, you will need to obtain permission directly
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Description and genomic characterization of Streptococcus symci sp. nov., isolated from a child’s oropharynxAbstractIntroductionMaterials and methodsSample collection and strain isolationStrain identification and gene sequencing of 16S rRNA, groEL, rpoB, and sodAPhylogenetic analysisMorphologic observation and optimal growth conditionsBiochemical and fatty acid methylester analysis and antibiotic susceptibility testBiochemical analysisFatty acid analysisAntibiotic susceptibility testing
Genomic DNA extraction, genome sequencing, and assemblyGenome annotation and analysis
ResultsPhylogenetic analysisPhenotypic characteristics and biochemical featuresGenomic propertiesGenomic comparative analysis between C17T and closely related species
DiscussionDescription of S. symci sp. nov.Author contributionsOpen AccessReferences